In a continuation of work on the receptor CHR3 which we previously cloned from c elegans, using a technique of soaking the animals with RNAi, we found that this had no effect on the larval stages, L2 and L3. Previous studies showed that RNAi injected into adult worms produced incomplete molting with the cuticle still attached to the animal. It may be that CHR3 may only be required for certain molts. To see if other genes may also be involved in molting we mutagenized 3,000 worms and found one mutation that caused a molting defect similar to that caused by a lack of CHR3 with incompletely detached cuticle. This gene has been localized to Chromosome 3 and hence is different than CHR3 which is on chromosome 1. We are in the process of localizing this gene more precisely and then from the sequence data available will be able identify it and study its mechanism of action. We had previously investigated expression and overexpression of CHR3. We now have additional data which shows that overexpression by the injection of large amounts of CHR3 DNA causes extra seam cells, additional alae and sometimes deformed embryos. These effects were seen with entire gene including its promoter and also when the LBD had been deleted. Future plans include a comparison of the promoter sequences of CHR3 in c briggsae and c vulgaris, somewhat distantly related nematodes to determine which sequences are conserved and hence likely to be major sites for control of gene activity. Other work which is under way will attempt to determine whether Ftz Fl which in the drosophila appears to be controlled by DHR3, is modulated byh CHR3. CHR3 is also homologous to ROR/RZR in mammals which affect transcription of some hemeodomain proteins. In c elegans egl-5 is responsible for tail development and we will see if CHR regulates its transcription. (Drs. Kostrouchova and Krause).In another investigation, we have studied the jelly fish, tripedalia cystophora. Last year we cloned a nuclear hormone receptor gene, jRXR from this species which is remarkably homologous to the vertebrate RXR gene. We also showed that it binds to certain promoter sequences in three jellyfish lens crystallin genes cloned by Joram Piatigorsky. Now we have shown that in a rabbit lens cell culture system, jRXR increased 4 to 10 fold the expression of luciferase which had been ligated to the crystallin promoter sequences. The effect of retinoids on this increase was not reproducible. No effect could be demonstrated in non lens tissues, such as NIH 3T3 cells or mouse embryonal carcinoma cells. JRXR could be detected by Western blotting in the rhopalia (eyes) of tripedalia. Surprising was the finding that with EM techniques, the main stain for jRXR was in pigment granules. Further studies with antibodies to different domains of the TR and RAR subfamilies gave positive results so we are currently attempting to clone additional members of the Nuclear hormone family of receptors from tripedalia. To search for endogenous retinoids in tripedalia, we examined larvae with help fromDr. Wiggert and were able tentatively to show the presence of all trans retinoic acid (jRXR binds only 9 cis retinoic acid). Further experiments are planned to get this straightened out. (Drs. Kostrouch and Piatigorsky).In another related study, we have examined nuclear hormone receptors in the mollusc Plactopecten magellenicus and agropectin irradians. Using RT-PCR we obtained the most consersed DBD regions of three different fragments about 150 bp in length. SC5 showed 76% identity with the orphan receptor TR2-11. SC7 showed 92% identity with CF1/USP, SC8 had 92% identity with seven-up and SC11 had 90% identity with E75. All of these are nuclear hormone receptors. USP is the drosophila homologue of RXR and with the EC receptor binds ecdysone. E75 is turned on by ecdysone and seven up is involved in photoreceptor cell development. It would appear that eye development is influenced by nuclear hormone receptors as is metamorphosis-- at least in drosophila. We are now attempting to get complete sequences of these genes and will study their function. (Drs. Carosa, Piatigorsky). - caenorhabditis elegans,epidermal cells, cnidaria, ciona intestinalis, nuclear hormone receptors